SE436036B - ANALOGY PROCEDURE FOR THE PREPARATION OF OXAZOLIDE INDION DERIVATIVES OF ANGLE ALKALOIDS - Google Patents

Description

_och att man därest R 7713586-1 lRß 5 7 'e'. \\ 11 '____ "T // 4'“ _ -R4 '1 19' U 8, 3 '___R5 12' áá \\ 13 __: § 9 » \\ 2 // 1a '_ Ü HI, \ / wxff /' w / '17 1: 1 [IV T' “- fi- O“ CHI5 H 1 o (II) BI // K \ 'BI 14 10 ___ * -N 7 '/ \ "2' 9 4 las / v 1% n Iz: T _ - _ CH2-CHa CH -O-- __ 6 ° \ /, å \ 1 / \ ß / 'R 1; N HO-f I 2 I R_ c-ocHß H O. . 2 3 4 5 _ - 6 N 1 in which R, R, R and R have the meaning given above and R is -OH or -OQ-CH5, is reacted with isocyanate of the formula RNCO in an inert OHRHÜÉKÛ solvent, where R has the above meaning, 7 is * Og-NHR possibly undertakes a hydrolysis to form a compound in which R? is -OH; and that the product is recovered in the form of free base or pharmaceutically acceptable salt thereof.

Formula I can also be written as the following formula III: 7713586-1 12: / _____.___ ° gr 15 '1, \ 0414' l I 1 \ “(4; \ §Å; wç¿: o + æom 14 1 IH Formula III H o 1 1 8 | I 1 ° // \\ I 10-- ~ u 7 l 1: 11 19 G \\.. \ Fl2 t {: -l2-: I // \} - - - - ons -ons rusk »- {: .U 1 /, §¿ \ j - W \ / ° \ 1 f; oÛl-czl-.zo 17 ln Is | 'IR” I Nå-R ¿4LLR9 than ll 0 in which R8 and R9 taken together are a single bond between Cu "and NB" and the other symbols have the meanings given above.

In the above formulas, the term "C 1 -C 6 alkyl" includes such alkyl groups as methyl, ethyl, n-propyl, isomethyl, n-butyl, isopropyl, sec-butyl and tert-butyl, and the term "C 3-8 alkenyl" includes allyl, metalallyl and crotyl.

Several naturally occurring alkaloids, which can be obtained from Vinca rosea, have been shown to be active in the treatment of experimentally induced disease states in animals. These include leurosine (U.S. Pat. No. 3,370,057), vincaleucoblastine (vinblastine), which will hereinafter be referred to as VLB (U.S. Pat. No. 3,097,157), leuroformin (Belgian patent 811,110), Ieurosidine (vinrosidine) and Ieurocristine ( vincristine) (both in U.S. Pat. No. 3,205,220), deoxy VLB "A" and "B", Tetrahedron Letters, 783 (1958), 4-desacetoxivine blastin (U.S. Pat. No. 5,954,773), 4-desaeeuoxy-3'- hydroxyvinblastine (U.S. Pat. No. 3,944,55R), leurocolombine (U.S. Patent 5,890,525) and vincadioline (U.S. Pat. No. 3,887,565). Two of these alkaloids, VLB and vincristine, are now marketed as drugs for the treatment of disease states, especially leukemias and related diseases in humans. Vincristine is the more active and useful agent for the treatment of leukemias, but '7713586-1 4 is also the less abundant of the antineoplastic alkaloids in Vinca rosea. These alkaloids are usually administered intravenously.

Chemical modification of the vinca alkaloids has been carried out to a limited extent. First, the same molecular structure is extremely complex, and chemical reactions that affect a specific functional group in the molecule without altering other groups are difficult to develop. Second, alkaloids lacking the desired chemotherapeutic properties have been extracted or prepared from Vinca rosea fractions or alkaloids, and a determination of their structure has led to the conclusion that these compounds are closely related to the active alkaloids. . Antineoplastic activity appears to be limited to very specific structures, and the chances of obtaining more active drugs by modifying these structures appear to be small. Among the successful modifications of physiologically active alkaloids have been the preparation of dihydric VLB (U.S. Patent No. 5,352,868) and the exchange of the acetyl group at C-4 (carbon atom 4 in the vinca alkaloid system - see the numerical structure below) for a higher alkanoyl group. or against unrelated acyl groups. (See U.S. Pat. No. 3,592,173.) Several of these derivatives are capable of prolonging the life of mice inoculated with leukemia P15}. One of the derivatives in which a chloroacetyl group replaced the C-4 acetyl group in VLB was also a useful intermediate for the preparation of structurally modified VLB compounds in which an N, N-dialkylglycyl group replaced the C-4 acetyl group in VLB (see U.S. Pat. U.S. Patent No. 5,387,001). C-3 carboxamide derivatives of VLB, vincristine, vincadioline, etc. have also been prepared and found to be active agents against tumors (Belgian Patent Specification 813,168). These compounds are extremely interesting because, for example, the 3-carboxamides of VLB are more active against Ridgeway's osteogenic sarcoma and Gardner's lymphosarcoma than the VLB from which they are made.

Some of the amide derivatives approach the activity of vincristine against these tumors. One of these amides, 4-desacetyl-VLB-C-3-carboxamide or vindesine, is currently used clinically in human trials and has been found to be active against certain leukemias. In humans, vindesine appears to have less neurotoxicity than vincristine.

One of the major medical problems is the treatment and care of neoplastic diseases in humans. There are a variety of these diseases and there is considerable specificity in compounds which have activity against various diseases. Minor modifications to the structure of the compound may affect both specificity and activity.

These new wine derivatives are welcome new weapons in the chemotherapist's arsenal, looking for new methods of treating neoplastic diseases.

Compounds of the above formula III can be generally described as derivatives of VLB, if R 7 is acetoxy, R 2 is methyl, H 3 is hydroxyl, R 4 is ethyl and R 5 is H, as derivatives of vincristine, if R is acetoxy, R 2 is formyl, H 3 is hydroxyl, Ru is ethyl and R 5 is H, as derivatives of desmethyl-VLB (also called desformylvincristine), if R 7 is acetoxy, H 2 is hydrogen, R; and Ru is hydroxyl and ethyl, respectively, and R 5 is H, as derivatives of leurosidine, if R 7 is acetoxy, R 2 is methyl, R 3 is ethyl, R is hydroxyl and R 5 is H, as derivatives of deoxy-VLB "A", if R 7 is acetoxy, H2 is methyl, H5 and H5 are hydrogen and R4 is ethyl, as a derivative of aeoxy-VLB "B", if R7, H2 and R5 are the same as 1 deoxy-VLB "A" but R5 is ethyl and R4 is hydrogen, as a derivative of leurosine, if R? is acetoxy, H2 is methyl, H5 is ethyl and R4 and R5 taken together form an α-epoxide ring, or derivative of leuroformin, the corresponding compound in which R2 = CHO. The compounds of the invention have been termed 5-spiro-5 "-oxazolidinedione derivatives of the particular alkaloid set forth above; for example, an oxazolidinedione obtained from VLB would be termed j-deskarbomethoxy -} - deshydrin® VLB-3-spiro-5" -oxazolide. din-2 ", 4" -dion. According to the above-mentioned name, a spiro compound is formed, in which the spiro atom is the carbon atom 3 in the vinca alkaloid system and the carbon atom 5 "in the oxazolidinedione ring system. In the name of compounds according to the invention, the term" 3-descarbomethoxy-3-deshydroxy "has been used to indicate carbomethoxy. and the hydroxy group at the carbon atom 5 has been replaced with (or incorporated into) the oxazolidine ring, however, in order to simplify the naming of the compounds of the invention, the term "5-descarbomethoxy-3-deshydroxy" will be avoided, as it is understood that the oxazolidine ring in each of the compounds has replaced the hydroxy and carbomethoxy groups at the carbon atom 3 of the vinca alkaloid, it will thus be appreciated that each name herein on an oxazolidinedione implicitly contains the term "5-aesxarbomethoxy-3-oesnydroxy".

Non-toxic acids which can be used to form pharmaceutically acceptable acid addition salts of the compounds of the invention include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid and the like. such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids and alkanedioic acids, aromatic acids, aliphatic sulfonic acids and aromatic sulfonic acids, etc. Such pharmaceutically acceptable salts thus include sulfates, pyrosulfates, bisulfates, monosulfates, bisulfates, bisulfates, bisulfates , dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, malarates, sebacates, sebacates -l, Ä- dioates, hexyn-1,6-dioates, benzoates, claw Benzoates, methyl benzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylene sulfonates, phenyl acetates, phenylpropionates, phenylbutyrates, citranes, methoxylates, maliculphonates naphthalene-1-sulfonates, naphthalene-2-sulfonates and similar salts. In the process of the invention, a dimeric indole dihydroindole alkaloid having an acetoxy group at C-4 and obtained either by extraction of the leaves of Vinca rosea or prepared semi-synthetically by chemically modifying such dimeric compounds or by coupling two monomeric indoles is reacted. dihydroindoids, etc., with an isocyanate RNco to give a β-carbamac intermediate, represented by formula III, in which R is H and H9 is -O-CH. These carbamates are relatively unstable at ordinary reaction temperature, such as 30-110 ° C, and rearranged to the spirooxazolidinediones represented by formula III, in which R8 and R9 are a single bond between G4 and NB. The stability of the 3-carbamate group depends to a large extent on the nature of the R-substituent at the carbamate nitrogen. For example, if R is / 5-chloroethyl, the 3-carbamate intermediate can be isolated from excess isocyanate in the reaction and the dimeric alkaloid (II) by chromatography and can be rearranged to a spirooxazolidinedione by heating. 5-Carbamates formed with methyl isocyanate are rearranged to the spirooxazolidinedione under standard reaction conditions. By carrying out the reaction at a relatively low temperature, the 3-carbamates can be isolated in most cases shortly after the isocyanate has been added. Heating for longer periods of time is not required. The isolated 3-carbamate intermediate is rearranged to a spirooxazolidinedione by heating to 30-11000 for several hours. The 5-carbamates of formula III are not only useful as intermediates in the synthesis of spirooxazolidinediones but also have activity against tumors themselves, as evidenced by their ability to inhibit the growth of transplanted tumors in mice. A second type of intermediate is often isolated from the reaction of an isocyanate and dimeric indole-dihydroindole alkaloid of formula II, especially if an excess of isocyanate is used and benzene is the solvent. This intermediate is a water-soluble precipitate comprising a complex of the vinca alkaloids and probably more than one molecule of the isocyanate per molecule of alkaloid. For example, if an excess of sheep chloroethyl isocyanate is reacted with VLB, the water-soluble complex that first forms forms a precipitate which is isolated by filtration.

A solution of the filtered complex in water gives the 3 "-Qß-chloroethyl) -spirooxazolidine dione after storage at room temperature for 16 hours. Other isooyanates behave similarly with VLB and other dimeric vinca alkaloids, but only some of the complexes (apparently dependent on the nature of the group R in the isocyanate RNCO) can be isolated at ordinary reaction temperatures.

In carrying out the above reaction scheme without isolating the carbamate intermediate (III), the vinca alkaloid (II) is dissolved in the form of the free base in an inert organic solvent such as benzene, and the desired isocyanate (RNCO) is added, usually in excess. The reaction mixture is crosslinked under reflux until the oxazazolidinedione formation is substantially complete and then cooled. Removal of the solvent and excess isocyanate by evaporation give the oxazolidinedione as a residue which can be further purified, for example by chromatography. Other inert solvents which can be used in the above reaction are methylene dichloride, chloroform, toluene and the like.

If a 4-desaoethyl alkaloid such as 4-desacetyl-VLB or 4-des-ethylvinycristine is used as starting material, the product of the isooyanate reaction is a 5-spiro-5 "-oxazolidine-2", 4 "-dione-4-carbamate (O- CO-NH) These 4-carbamatoxazolidinedione derivatives are also active agents against tumors, as well as the intermediates for the preparation of compounds in which R 1 is OH or finally acetoxy.

Compounds of formula III in which H7 is OH are prepared by acidic hydrolysis of compounds of formula III in which R7 is acetoxy, e.g. with hydrochloric acid. These compounds, in which R 7 is OH, can be reacted with an isocyanate (RNCO) to give compounds in which RT is O-C-NH-R by simple reaction in an inert solvent, preferably under gentle heating. These 4-carbamates are also prepared by using in the above reaction scheme a compound of formula II, in which R is OH, as starting material.

Pharmaceutically acceptable acid addition salts of the} -spiro-5 "-oxazolidine-2", 4 "-dione derivatives of these vinca alkaloids, represented by formula III, are prepared by mixing a solution of the free alkaloid base with one mole of the desired non-toxic acid in an inert solvent. If the salt is soluble, it is recovered by evaporation of the solvent in vacuo. If the salt is insoluble in the reaction medium, it precipitates and can be recovered by filtration.

Some of the inorganic salts are prepared by a slightly different procedure. The sulphate salt is prepared, for example, by dissolving a free base of formula III above in a minimum amount of a polar organic solvent, such as ethanol, after which the pH of this solution is adjusted to about 4.0 by dropwise addition of 2% aqueous tig sulfuric acid in ethanol. The sulphate salt is recovered by evaporating the solvents to dryness. The hydrochloride salt can be prepared in a similar manner by adding an alcoholic solution of HCl to an alcoholic solution of the free base. The acid addition salts prepared in the above manner can be purified by such well known techniques as chromatography or recrystallization.

As mentioned above, the compounds of the invention are also useful as intermediates for the preparation of other vinca alkaloids having action against tumors. Treatment of the oxazolidinedione (I) with bases gives a C-3 carboxamide derivative, which usually lacks the acetyl group at C-4. Reaction of, for example, VLB-3 "-methyl-3-spiro-5" -oxazolidine-2 ", 4" -dione with aqueous alkali gives 4-desacetyl-VLB-C-3-N-methylcarboxamide. The C-3-carboxamide derivatives of vinca alkaloids, such as VLB, vincristine, leurosidine and deoxy-VLB "A" and "B" are active agents against tumors in animals, as stated in Swedish patent 7770059-2. The C-3 carboxamides of leurosine and of leuroform, which alkaloids are within the scope of formula II of this patent, are also active agents against tumors transplanted into experimental animals. The starting materials of formula II above are isolated either from the leaves of the plant Vinca rosea, such as VLB, vincristine, leurosidine, leurosine, leuroformin, deoxy-VLB "A" and "B", or, if R in formula II is OH is prepared by acid hydrolysis of the corresponding compound in which R 6 is O-9-CH 3. The 4-desacetyl compound can be re-acetylated if desired at C-4 by the method of Hargrove, Lloydia, 21, p. 540 (1964) or by acetylation under mild conditions using one mole of acetic anhydride. Starting materials represented by the formula II, in which R2 is H or formyl, with the exception of vincristine, N-desformylleurocristine, N-desformylleuroformine and leuroformin, all of which are obtained from the leaves of Vinca rosea, are prepared as follows: . The 1-methyl group in deoxy-VLB "A" or "B" etc. can be oxidized with chromium oxide in glacial acetic acid at -6000 to give a mixture of compounds in which H2 is H or formyl, all according to the procedure disclosed in U.S. Patent No. 3,899,493. The compounds in which H 2 is H may be formylated, or compounds in which R 2 is formyl may be deformylated.

The following examples are intended to further illustrate the invention without, therefore, limiting the same.

Example 1.

Preparation of VLB-3 "-methyl-3-spiro-5" -oxazolidine-2 ", 4" -dione.

A solution was prepared containing 2.84 g of free VLB base in 40 ml of anhydrous benzene. 15 ml of methyl isocyanate were added and the mixture thus obtained was heated to reflux temperature for about six hours. The reaction mixture was cooled and the volatiles were removed by evaporation. The residue comprising VLB-3 "-methyl-3-spiro-5" -oxazolidine-2 ", 4" -dione formed in the above reaction, was purified by chromatography over 200 g of silica (activity I).

The chromatogram was developed with one liter of 4% methanol in benzene, followed by 2.5 l of 6% methanol in benzene, which eluted VLB-3 "-methyl-3-spiro-5" - <> xaz611a1n-2 ", 4 "-d1on. Yield = 0.9764 g; moiexyl ion week at 835; nmr d'vid 0.17, 0.88, 2.0, 2.60, 2.74, 2.78, 2.81, 3.08 (ny Nc fl š), 5.58, 5.60, 3, 79, 4.52, 5.18, 5.41, 5.88, 6.09, 6.64; mass spectrum peak at 849 (transmethylation), 855, 817, 804, 776, 650, 543, 494, 355, 167, 154, 149, 135.

Using the above procedure, 270 mg of free VLB base was reacted with n-butyl isocyanate to give VLB-3 "-n-butyl-3-spiro-5" -oxazolidine-2 ", 4" -dione (41 mg of purified material) . Mass spectrum peaks at 891, 877, 859, 857, 856, 819, 818, 792, 747, 650, 545, 555, (512), 581, 580, 555, 541, 525, 295 (188), 154, 149 , 155 00h 156, 122, 121, 107. Nmr 1 deunerokioroform, ofvid 0.66, 0.92, 1.5-1.7, 1.99, 2.07, 2.81, 5.61, 5, 79, 5.17, 5.43, 5.87, 6.09, 6.61. 98.9 mg of free VLB base was reacted with allyl isocyanate to give VLB-3 "-allyl-3-spiro-5" -oxazolidine-2 ", 4" -dione (31.3 mg of purified material). Mass spectrum ion peaks at 875, 861, 843 and 844, 830 and 831, 802 and 803, (784), 650, 543, 520, 380 and 381, 355, 295, 273, 242, 154, 149, 143, 36, 135, 122, 121, 107. Infrared spectrum 1 chloroform; peaks at 5440, 1810, 1740, 1610 cnfl. Nmmd at 0.66, 0.88, 1.98, 2.41, 2.30, 3.57, 5.50, 3.79, 4.14, 5.1-5.3, 5.43, 5.7-5.95, 6.08, 6.63. 7713586-1 Example 2.

Preparation of vincristine-5 "-methyl-5-spiro-5" -oxazolidine-2 ", 4" -dione.

Free vincristine base obtained from 158.5 mg of vincristine sulfate by standard procedures was dissolved in about 15 ml of anhydrous benzene. About 2.5 ml of methyl isocyanate was added to the vincristine solution, and the resulting mixture was heated under reflux for 6 hours and then kept at room temperature overnight, followed by another 4 hours of heating under reflux. The volatiles were removed to give a residue weighing 159.6 mg which was soluble in methanol but insoluble in methylene dichloride. Preparative thin layer chromatography using a solvent system 5: 1 ethyl acetate-ethanol gave four bands, the third of which, in polarity, was vincristine-5 "-methyl-5-spiro-5" -oxazolidine-2 ", 4" -dione (26.5 mg purified material) with the following physical properties. Mass spectrum peaks at 865, 849, 851, 818, 806, 790, 751, 708, 647, 650, 635, 480, 595, 569, 555, 285, 270, 268, 185, 171, 168, 154, 141, 156, 122, 121.

Infrared spectrum in chloroform; peaks at 5440, 1805, 1750, (and 1745), 1660 cm_l. Nmr in deuterochloroform; dxvid 0.59, 0.88, 1.55, 1.68, 2.74, 2.78, 5.01, 5.65, 5.87, 4.41, 4.68, 5.20, 5 , 90, 6.81, 6.91, 8.12, 8.78. Example 2.

Preparation of 1-desmethyl-4-desacetyl-4- (N-methylcarbamoyloxy) -VLB-5 "-methyl-5-spiro-5" -oxazolidine-2 ", 4" -dione.

Using the procedure of Example 1, 21.7 mg of 1-desmethyl-4-desacetyl-VLB was reacted with 5 ml of methyl isocyanate in about 12 ml of anhydrous methylene dichloride at reflux temperature. 1-Desmethyl-4-desacetyl-4- (N-methylcarbamoyloxy) -VLB-5 "-methyl-5-spiro-5" -oxazolidine-2 ", 4" -dione, which was formed in the above reaction, was isolated according to the procedure in Example 1 and purified by preparative thin layer chromatography using a methanol solvent system (5.5 mg purified product).

The compound had the following physical properties. Nmr (in deuterochloroform); d "at 2.75, 2.79, 3.08, 3.59, 3.76, 3.84, 4.76, 5.35, 5.19, 5.86, 6.26, 6.68 Infrared spectrum in chloroform showed a new peak at 1.88 cm -1, as well as increased absorption at 174 cm -1 and at 115 cm -1, compared with the starting material. Mass spectrum ion peaks at 856, 854, 779, 777, 568, 256, 241, 215, 155 and 149.

Example 4.

Preparation of VLB-5 "- (β-chloroethyl) -5-spiro-5" -oxazolidine-2 ", 4" -dione.

Using the procedure of Example 1, 1.620 g of VLB was reacted with 15 ml of β-chloroethyl isocyanate in 100 ml of anhydrous benzene. The reaction mixture was stirred for 16 hours at room temperature followed by 2 hours of heating under reflux. A precipitate containing a water-soluble complex of VLB and f-chloroethyl isocyanate was separated and recovered from the cooled solution by filtration. The precipitate (1,692 g) was dissolved in about 50 ml of water and the aqueous solution was stirred at room temperature for about 16 hours. The solution was then basified by the addition of dilute sodium hydroxide and VLB-5 "- ([9-chloroethyl) -5-spiro-5" -oxazolidine-2 ", 4" -dione, which was formed in the above reaction and was insoluble in basic solution. , was separated and extracted into methylene dichloride. The methylene dichloride extracts were combined and the solvent was removed by evaporation. The residue containing VLB-5 "- ([6-chloroethyl) -5-spiro-5" -oxazolidine-2 ", 4" -dione was purified by chromatography over 150 g of silica (activity I) (1.127 g of purified material) .

The chromatogram was developed with 6% methanol in benzene. The compound had the following physical properties; mass spectrum ion peaks at 885, 847, 816, 789, 650, 592, 591, 543, 505, 485, 451, 555, 295, 154, 156, 155, 122, 121, 107. Infrared spectrum (in chloroform) peaks at 5650, 5570 , 5440, 1805, 1745, 1610 cm -1. Nmr in deuterochloroform, OF at 0.67, 0.88, 2.01, 2.81, 2.85. 5.61, 3.79, 3.87, 5.19, 5.44, 5.89, 6.11 and 6.65.

Example 5.

Alternative Preparation of VLB-5 "-Qβ-Chloroethyl) -5-spiro-5" -oxazolidin-2 ", 4" "-dione and VLB-5-N- (β-chloroethyl carbamate. - A solution was prepared containing 481.2 mg of free VLB base in 14 ml of anhydrous benzene, 7.0 ml of chloroethyl isocyanate was added, a precipitate formed immediately, the reaction mixture was heated to reflux for about 16 hours, the benzene was removed by evaporation in vacuo and the residue was subjected to preparative thin layer chromatography on silica using a solvent system 5: 1 ethyl acetate-methanol Two non-separable moving bands were isolated together (with a total yield of 579.4 mg) and chromatographed again. A second chromatography using the same solvent system of 5: 1 ethyl acetate-methanol with only 47 mg of the two-component mixture per preparative plate resulted in a separation into VLB (55% (β-chloroethyl) -5-spiro-5 "-oxazolidine-2", 4 "-dione (147 mg) and VLB-5-N- (β-chloroethyl) carbamate (69 mg). This senane compound had the following physical properties; mass spectrum ion peaks at (881), 855, 821, 790, 763, 692, 591, 543, 480, 409, 353, 295, 293, 283, 281, 278, 245, 188, 154, 149, 155. Infrared spectra in chloroform exhibited maxima at 5645, 5575, 5450, 5405 (new), 1750, 1675 (new), 1610 cm_l.

Nmr in deuterochloroform; 3.60, 3.77, 5.32, 5.53, 5.84, 6.09, 6.60, 10.0 (broad). 7713586-1 12 Example 6.

Preparation of 4-desacetyl-VLB-5 "-methyl-5-spiro-5" -oxazolidone-2 ", 4" -dione. 99.7 mg of VLB-5 "-methyl-5-spiro-5" -oxazolidine-2 ", 4" -dione was refluxed in 0.5 N aqueous hydrochloric acid for about 1.5 hours.

The aqueous solution was basified and 4-desacetyl-VL-5 "-methyl-5-spiro-5'-oxazazolidone-z" AW-dione, which is insoluble in basic solution, precipitated and extracted into methylene dichloride. The methylene dichloride extracts were combined and the solvents removed in vacuo. preparative thin layer chromatography EVER silica gel using a solvent mixture of 5: 1 ethyl acetate-methanol to develop the chromatogram. The main band comprising 4-desacetyl-VLB-5 "-methyl-5-spiro-5" -oxazolide-2 ", 4" -dione (52.5 mg of purified material). The compound was a single spot material in three other chromatographic systems.

It had the following physical properties: mass spectrum ion peaks at 807, 793, 763, 762, 749, 734, 590, 493, 452, 408, 555, 295, 268, 167, 171, 154, 149, 145, 155, 122, 121, 107. Infrared spectrum 1 chloroform; maxima at 5580, 5440, 1815, 1755, 1615 cm_l. Nmr in deuterochloroform; cr at 0.80, 0.88, 2.70, 2.81, 2.90, 5.04, 3, s9, '3.76, 3.92, 4.20, 5.60, 5.88 .

Example 7.

Preparation of 4-desacetyl-VLB-5 "-methyl-5-spiro-5" -oxazolidine-2 ", 4" -dione-4- (N-methyl) carbamate.

A solution was prepared containing 504 g of 4-desacetyl-VLB in 10 ml of anhydrous benzene. 5 ml of methyl isocyanate were added and the solution thus obtained was refluxed for about 6 hours. The solvent was removed by evaporation in vacuo to leave a residue weighing 444.4 mg. Thin layer chromatography indicated that two materials were present in the residue, and these were separated by preparative thin layer chromatography using a 5: 1 ethyl acetate-methanol solvent system. There was obtained purified 4-desacetyl-VLB-5 "-methyl-5-spiro-5" -oxazolidine-2 ", 4" -dione-4- (N-methyl) carbamate having the following physical properties: mass spectrum peak at 850, 807, 795, 775, 762, 754, 452, 555, 154 and 155. Ultraviolet spectrum in ethanol; peaks at 215 and 268 with approaches at 290 and 298 pm. Infrared spectrum (in chloroform); maxima at 5700, 5460, 1816, 1745, 1600 cmfl. Nmr in deuterochloroform; GV at 0.67, 0.88, 2.78, 2.81, 5.07, 5.60, 5.70, 5.0, 5.25, 5.50, 5.84, 6.08 and 6.05. This compound showed metakin inhibitory activity in a standard mitosin inhibition assay at concentrations as low as 10-3 pg / ml.

The residue was subjected to Example B.

Preparation of 4-desacetyl-VLB-5 "-β-chloroethyl-3-spiro-5" -oxazolidine-2 ", 4" -dione. 25 ml of dry methanol saturated with hydrogen chloride at 000 was added to 96.9 mg of VLB-3 "78-chloroethyl-3-spiro-5" -oxazolidine-2 ", 4U-dione. The mixture was allowed to warm to room temperature and stirred for 18 hours. The solvent was evaporated in vacuo. Water was added to the rest along with ice. The solution was basified with ammonium hydroxide and extracted four times with methylene dichloride. The methylene dichloride extracts were combined and the solvents evaporated in vacuo. The residue (88.6 mg) was subjected to preparative thin layer chromatography on silica gel using a solvent mixture of 3: 1 ethyl acetate-methanol to give the chromatogram. The band showing Rf = 0.4 was eluted with a yield of 14 . Physical properties: mass spexcrumuoppar at 841 (stamjon), 816, 782, 651, 545, 555, 295, 175, 167, 154, lÄ9, l} 5, l25. "gnempel 2.

Alternative preparation of 4-desacetyl-VLB-3 "V8-chloroethyl-3-spiro-5" -oxazolidine-2 ", 4" -dione. 25 ml of 16-chloroethyl isocyanate was added to 9.86 g of free VLB base in 200 ml of dry benzene. The reaction mixture was stirred overnight at room temperature and then refluxed for 2.5 hours. The precipitate was filtered off. 5.15 g of filtrate was dissolved in 300 ml of 0.1 N aqueous hydrochloric acid and boiled under reflux overnight. The reaction mixture was cooled and basified with ammonium hydroxide and extracted with methylene dichloride. The extracts were combined and the solvent was evaporated in vacuo to give a residue of 2.80 g. The residue containing H-desacetyl-VLB-β-chloroethyl-3-spiro-5 "-oxazolidine-2", U, "-dione The chromotogram was developed with benzene with an increasing methanol gradient, the yield was 192.0 mg Physical properties: mass spectrum ion peaks identical to those in Example 8. Nmr in deuterochloroform Åvid 0.81, 0.88,?, P §, 2.7l, 2.99, 3.60, 5.76, 5.79, 3.93, 5.61, H, 88, 6.00 and 6.63.

Example 10.

Preparation of salts. Salts of the oxazolidinediones (I above) were prepared as follows: The free base is dissolved in the least possible amount of ethanol, the pH is lowered to 0, 0 É 0.2 by dropwise addition of 2% sulfuric acid in ethanol. The pH is determined by taking a two-drop sample, diluting these drops to 1 ml with water and determining the pH using a pH meter. The acidic ethanolic solution is evaporated to dryness. The residue, which comprises the sulphate salt, can be recrystallized from methanol or ethanol if desired.

Example ll. Preparation of 4'-deoxy-leurosidine-3 "-methyl-3-spiro-5" -oxazolidine-2 ", - H" -dione. U 215.7 mg of deoxyleurosidine (free base) was boiled for 0.5 hours under reflux in 3 ml of dry toluene and 2 ml of methyl isocyanate. In thin-chain chromatography, a blue spot representing an RF value similar to that of the starting material was obtained as a characteristic of the product. After evaporating the solvent to leave a dry residue, it was chromatographed on a thin layer chromatography preparation using a 9: 1 methylene dichloride / methanol solvent system.

The amount of product isolated was 59.0 mg / l Mass spectrum: 8.19 (original ion), 817, 787, 759, 757, 689, 633, 60, 527, 525, 11911, 580, 538, 1115, 138, 135, 1211

NMR (cnciz fi o "o, 61n ;, o, 811t, 1.98 oAc, _3.07 _NCH3, 2.80 NCHB, 3.59 COZCH3, 379 CH5OAr, 5.82 m, 6.07, 6.57, Infrared spectrum (CHCl 3) 3680, 3U70, 1815 (characteristic of oxazolidinedione ring fi h 17ü0 cm_1.

The product was transferred to sulfate salt in the usual manner.

Example 12.

Preparation of H'-deoxy-leurosidine-3 "- (Fß-fetyl) -3-spiro-5" -oxazolidin-2 ", 4" -aion. 89.5 mg of deoxy-leurosidine (free base) were dissolved in 3 ml of dry toluene together with 2.5 ml of chloroethyl isocyanate and heated for 1 hour to 8000. Upon evaporation of the solvent, an oil was obtained. The product was chromatographed on 20 g of Woehlm silica gel, activity I, first prepared and eluted with 150 ml of 1: 1 ethyl acetate / methylene dichloride and 10% methanol; then with 100 ml of ethyl acetate / methylene dichloride with 15% methanol; then with 100 ml of ethyl acetate / methylene dichloride with 22% methanol; and then with 100 ml of ethyl acetate / methylene dichloride with 33% methanol. Hü, 9 mg product, was isolated.

Infrared spectrum (CHCl 3): 3680, 3550, 3H70, 1820, 1755, 1735, 1715.

Mass spectrum: 867 (original ion), 865, 836, 831, 809, 800, 773, 573, 1167, 1151, 390, 338, 252, 2014, 1711, 138, 135, 1211.

The product was transferred to sulfate salt in the usual manner.

Example 13

Preparation of leurosin-3 "-methyl-3-spiro-5" -oxazolidine-2 ", U" -dione.

To 66.8 mg of leurosine in 1-2 ml of dry toluene was added 1.0 ml of methyl isocyanate, and the reaction solution was heated for 27 hours under reflux. The solvent was evaporated, leaving a solid, resinous substance weighing 77.0 mg. It was chromatographed on a thin layer chromatography preparation using a 5: 1 ethyl acetate / ethanol solvent system. The amount of product isolated was H, 7 mg.

Mass spectrum: 807 (transmethylation), 835 (original ion), 802, 69ü, 6u8, 635, BM1, 509, furnace, H51, H3h, 589, 369, 3h5, 295, 282, 221, 171, 166, ing, ins, 155, 120-2, 106, 107.

Infrared spectrum (CHCl3) 3680, 3060, 5300, 1810, 1755, 1690.

Example lü.

Preparation of 1-formyl-1-desmethyl-leurosine-3 "-methyl-5-spiro-5" -oxazolidine-2 ", 4" -dione.

A solution of 89.0 mg of formylleurosine in 2.0 ml of dry toluene and 2.0 ml of methyl isocyanate was heated for 21 hours to 8000. The solvent was evaporated. The residue was purified by thin layer chromatography using a 3: 1 ethyl acetate / ethanol solvent system. U, ü mg of product was isolated.

Infrared spectrum (CHCl 3): 5670, 3U50, 1810 (characteristic of oxazolidinedione), 1730, 1670 (formyl).

Mass spectrum: 861 (transmethylation), 853, 805, 756, 730, 665, 5H7, SOU, H25, E98, 387, B69, 553, 323, 282, 252, 1U9, 156, 155, 122, 121, 114, 107 .

The compounds of the invention have been shown to be active against transplanted tumors in mice in vivo and to prevent further development of Chinese hamster avovary cells before metaphase, when the cells are kept in tissue culture. When demonstrating the activity of the drugs of the invention against transplanted tumors in mice, a protocol was used which included administration of the drug, usually by the intraperitoneal route, at a given dose for 7-10 days after inoculation with the tumor.

The following Table 1 gives the results of several experiments in which mice with transplanted tumors were successfully treated with a compound of the invention. In the table, the name of the association is given in column 1. in column 2 the truncated tumor, in column 3 the dose or interval of dose and the number of days the dose was administered, in column 4 the delivery rate and in column 5 the percentage inhibition of the tumor growth or the percentage prolongation of survival. (HOS is an abbreviation for Ridgeway's osteogenic sarcoma; GLS for Gardner's lymphosarcoma; Pl534 (J) and Pj88 are leukemias; CA 755 is an adenocarcinoma and B16 is a molanoma.) _ TK davn. márffw., k .. _. »W fl ønmo .Tmv ïøfmm Û f u." Oáèí, ÉÜD wmæl fi w mH ca X mo | vo M ^ wUcm> wH woo fi Om mx mm Änvvmm fl m Jwmš »w fl uumø m? 3512 Û 5 x máffo .Bm æNm | mm _ mlm mnwmw _ WH o fi x oH | m> .o mmßmu. ol | ~ WH l vn fi n | .. 1 _. | mr Of lm O mo WHO Q fl NWuMMHÜW woo fl lmm Om. m K wlm> w> | om IN. | .w NH mw O nu .. O u: WÜQICOHÜISÉ-.SN _ ^ | m «U fi HonmHo | = m: ohHQw | m I H .. wwhOlrv fl l fl wlz fi lmå .wcafm Möïlow _ _ Û må w máå ... mä no fl n u ofl x oxjm. fm: Hæumffff fi um fi ï fi hvwommwønw: u _.. .. ^ Ü fi o fl hovdmnmx fl mnwënzv La I 'vn ß-lm. woo fi Om oa X vlm m fl w WOOH. Om w un vlm 33 H 3 x wfm UM aM mM 31/3 . 0A xmv \ .Hw> .o uw fi ufucmo MV å .. n. _ 1..

Awdïüsmi a wu fl AH of X m Olm O w fl m DHGMQMMHSW Ulm r url v r _. xßw> m uu fl nvcmo + _. v .w fl ï fi lmm _ wï m xmmï fi loï fi o fl m fl q 1 .o. . V "mN-.mn wOOH ... h _ .w. ._ 8 r.. Man, 5 _ _ _ upwwwJl ..: Nuu fi v fl afowmxouím 3 f.) Q faí _ | o: .fuw | M: .m. .mnm__h «__ m | mH> fl nm 41 o .Ãuømhbnfn fi rfn ... hmw fl wmnmm fifl wß Gwàu N uuim fi 1 ::. - nl.> m cc fl nmøm fl nmw nm flfl m. .won.: i .E ucærmßmm nl:: o øm.

H .fifl wßm fi .mU: m> wH »m> m uw flfi vcmo 7713586-1 Eom øwns fi ww mmøëmvwß sno ow www mm mucmamwpæou mU> wH Ldwø mm @ fi umømc> mHmw> w> m .cwcmHLmm æoo fi xHuw 17 UHmwUmc> m fl mm> ©> m mc fl cwnm fl nnm mm flfl m co fl u fl ß fl ß flfi uümoohm. pn fl wamwmßma flfl ß Gunn K ux \ mE ^ .w »» o @ v H H fl wnma m fi m. Åhv fi mm fl m muw m fi ü mdw m fl w 47.11,4: VTI-JL .pmu fi u fl xou> æ mUmxmmuoLæ @ fifi mmmømv mwm fi mwc fl swn fl sc fl mNm% w .vmxmmnmw> Ü Ü L ß Ü ß Ü ß ß Ü ß ß. | =:. = N | m fi øHHoN «xo | = m | o @» fl @ w «m | ^ H>» @ »oHx | Qv» = m «xc fififl moàäm fl n fi kowwlz mo« m1 = f. = N | GHø4MoNmMo | "m | oh fl mw | M | ^ Hhumho fi x -mV- = m | mq> | Hm »@@« wmu »@ GOMÜI: ä .. n N | u fl U fl moNmMo | = m | oh fl om | n | ~ m» ms | = n | a fl pmwhxuH > n @ fl n | f1. = m | c fl ø fifl ø ~ mxo \ = m1om fl mw | m | fl mHHm | = M | mA> .QOHÜIš .I n: Nl flflfl | H fl ommxo | = m | om «ow | n | fi hpmE |: n | As shown in the above table, the oxazolidinediones according to the invention are active in oral administration, this oral activity being completely unexpected, since the commercially available drugs VLB and vincristine do not exhibit this degree of oral activity against the same tumors induced experimentally in animals. For example, VLB-3 "-methyl-3-spiro-5" -oxazolidin-2 ", #" -dione and its j "-Q @ -chloroethyl) analog 100% orally inhibitory activity against GLS, ROS and CA755 at doses of 6-7 and 3-6 mg / kg, while VLB and vincristine are only marginally active, if at all, at doses of 2 and 2-4.5, respectively. mg / kg and are usually toxic at higher doses. However, parenterally (via the intraperitoneal route), comparable tumor inhibition or prolongation of life is obtained at doses of 0.5-0.75 mg / kg for the oxaolidinediones and at doses of 0.4-0.5 mg / kg for VLB and 0 , 2-0.25 for vincristine. When using the novel okazolidinediones according to the invention as anti-tumor agents in mammals, the administration can take place either by parenteral or oral route. For oral dosing, a suitable amount of a pharmaceutically acceptable salt of a base of formula III is mixed, which salt is formed with a non-toxic acid, with starch or other excipients, and the mixture is placed in telescopic gelatin capsules, each containing from 7 Similarly, a pharmaceutically acceptable salt may be mixed with starch, a binder and a lubricant, and the mixture may be compressed into tablets each containing from 7.5 to 50 mg of salt. . The tablets may be provided with a cutter, if lower or divided doses are to be used. In parenteral administration, the intravenous route of administration is preferred, although with smaller mammals such as mice the intraperitoneal route of administration is used.

For parenteral administration, isotonic solutions containing 1-10 mg / ml of a salt of an oxazolidinedione of formula III are used. The compounds are administered in amounts of from 0.01 to 1 mg / kg and preferably from 0.1 to 1 mg / kg of the mammalian body weight once or twice a week or every other week, depending on both the activity and the toxicity of the drug. An alternative method of calculating a therapeutic dose is based on body surface area: A suitable dose of 0.1-10 mg / m 2 mammalian body surface area every 7 days or days. '

Claims (1)

1. 7713586-1 19 P a t e n t k r a V Process for the preparation of a dimeric indole dihydroindole dione compound of the formula 42min »'., 4 f --- c-0-cH @ (I) 1 * | I n H I o I u [U '10 - ~ ---: // \\ I7 I; H w / lï / Us i15 \ fi lš --'- är '' _ _ "cHz-CHZE» CH -o-- 'B ~ 2, “----- R7 a \\\, / 1B \ 3 / C 1 -C 10 alkyl, C 3 -C 6 -alkenyl or -CH 2 -CHQX where X is Br or Cl; R 2 is ; H, -c fl or -cHo; one of R3 and R taken separately is -H or -OH and the other -C2H5 and H5 is H; or Ru and H5 together form an epoxide ring and RB is -C2H5; and R? is -OH, -O-O-NHR or -O-ε-CH 3, characterized in that a dimeric indole dihydroindole of the formula '7713586-1 s' Ra 11', 7L ~ __§I /: * \ '"' _ -Rá, / I / \} ï7 ?. ' v? "Rs L f, C_O_CHS (II) sH: å 8 I I I 14 1 ° ___" _: // \\ fl7 1 0 11 19 s 4 \ laïz / \ /. CH3_0__ lä Ü _I2: I: _; e ~ ~ CH2-cHs - \ 1; / 18 \ '? / H§¿; / R2 o-ocHs no 6 H and H5 have the meaning given above and R is in which H2, H5, R -OH or -OE-CH5.0 are reacted with an isocyanate of the formula RNCO in an inert organic solvent, where R has the meaning given above, and that R is -O-F-NHR possibly performing a hydrolysis to form a compound in which R? is -OH; and that the product is recovered in the form of free base or pharmaceutically acceptable salt thereof.